Cryptosporidium species, including C. parvum and C. hominis, are protozoan parasites that present a health threat particularly to immunocompromised individuals and young children. These organisms are also potential bio-terrorism agents that could incapacitate large populations. Current treatment options are limited. Commonly used antiparasitic drugs are ineffective, the only FDA-approved drug is poorly efficacious, and vaccines are unavailable. Genomic analysis has revealed that Cryptosporidium species rely on inosine 5'- monophosphate dehydrogenase (IMPDH) for producing guanine nucleotides and, hence, survival. It also appears that the parasite obtained its IMPDH gene from an ?-proteobacterium by lateral transfer, so C. parvum IMPDH (CpIMPDH) is highly diverged from the human orthologs. Therefore, selective CpIMPDH inhibitors may provide an effective strategy for the treatment of cryptosporidiosis with minimum toxicity to the patient. To date, several structurally distinct classes of CpIMPDH inhibitors have been identified that have demonstrated excellent enzymatic potency, selectivity over human IMPDH, anti-parasitic activity in a cell culture model and, for one compound series, in vivo efficacy in an acute cryptosporidiosis mouse model. The overall goals of this proposed study are to further refine CpIMPDH inhibitors to achieve efficacy in both acute and chronic mouse models of cryptosporidiosis to support the hypothesis that selective CpIMPDH inhibition is a viable treatment strategy for this protozoan infection, provide guidance for the research community with respect to optimal compound properties for in vivo efficacy and provide further pre-clinical validation of CpIMPDH as a molecular target. These goals will be achieved by pursuing three specific aims: 1) design, synthesis and in vitro evaluation of CpIMPDH inhibitors in assays of CpIMPDH inhibitory potency and selectivity, a C. parvum cell culture infection model and ADME properties (including mouse and human intestinal and liver microsome stability, cellular uptake, efflux and toxicity, and UGT-mediated glucuronidation); 2) assess the in vivo pharmacokinetic and acute toxicity properties of CpIMPDH inhibitors, including in vivo models of oral absorption and tissue distribution in both non-infected and C. parvum infected mice; 3) evaluate optimized CpIMPDH inhibitors in acute and chronic animal models of cryptosporidiosis assessing fecal oocyst excretion and gastrointestinal gross and histopathology. In addition, the impact of CpIMPDH inhibitors on commensal bacteria populations will also be examined to more fully evaluate pharmacodynamics effects, since many bacteria have IMPDHs that are similar to CpIMPDH. This project will also develop strategies for limiting drug distribution to the gastrointestinal tract. Since this strategy could be applicable to other gastrointestinal diseases, such as irritable bowel disease and colon cancer, this work has broad implications beyond the treatment of cryptosporidiosis.